Bottom Line:
Using an iterative assignment and structure calculation protocol, a large number of distance restraints was extracted from (1)H/(1)H mixing experiments recorded on a single uniformly labeled sample under magic angle spinning conditions.The calculated structure has a coordinate precision of 0.6 A and 1.3 A for the backbone and side chain heavy atoms, respectively, and deviates from the structure observed in solution.The approach is expected to be applicable to larger systems enabling the determination of high-resolution structures of amyloid or membrane proteins.

ABSTRACTHigh-resolution solid-state NMR spectroscopy can provide structural information of proteins that cannot be studied by X-ray crystallography or solution NMR spectroscopy. Here we demonstrate that it is possible to determine a protein structure by solid-state NMR to a resolution comparable to that by solution NMR. Using an iterative assignment and structure calculation protocol, a large number of distance restraints was extracted from (1)H/(1)H mixing experiments recorded on a single uniformly labeled sample under magic angle spinning conditions. The calculated structure has a coordinate precision of 0.6 A and 1.3 A for the backbone and side chain heavy atoms, respectively, and deviates from the structure observed in solution. The approach is expected to be applicable to larger systems enabling the determination of high-resolution structures of amyloid or membrane proteins.

pone-0002359-g002: High-resolution 3D structure of KTX determined in the solid phase.Stereo view of the 20 lowest-energy structures are shown.

Mentions:
Previously, 15 long-range, 7 medium-range and 6 short-range correlations could be assigned [11]. Using the above described semi-automated approach a total of 260 1H-1H distance correlations could be assigned unambiguously (Figure 1CD and Table 1). 62 of these were long-range, 33 medium-range and 165 sequential. The 3D solid-state structure of KTX that was calculated from the 260 distance restraints and 58 dihedral angle restraints is shown in Figure 2. The resulting ensemble of KTX structures tightly converged with a coordinate precision of 0.6 Å and 1.3 Å for backbone and side chain heavy atoms, respectively. Backbone and most side chains had a well-defined orientation except the N- and C- terminal residues and Asn30 located in the loop connecting the second and third β-strand of KTX (Figure 3). The high-resolution solid-state structure of KTX deviates by 2.4 Å from the backbone conformation (PDB code: 1XSW) obtained on the basis of 28 manually assigned distance restraints[11], which deviates by 2.7 Å from the solution structure. The most pronounced deviation between 1XSW and the high-resolution structure was observed at the N-terminus, where four residues were rotated by about 50°, such that the first β-strand was straight and not bent as seen in the high-resolution structures (Figure S1).

pone-0002359-g002: High-resolution 3D structure of KTX determined in the solid phase.Stereo view of the 20 lowest-energy structures are shown.

Mentions:
Previously, 15 long-range, 7 medium-range and 6 short-range correlations could be assigned [11]. Using the above described semi-automated approach a total of 260 1H-1H distance correlations could be assigned unambiguously (Figure 1CD and Table 1). 62 of these were long-range, 33 medium-range and 165 sequential. The 3D solid-state structure of KTX that was calculated from the 260 distance restraints and 58 dihedral angle restraints is shown in Figure 2. The resulting ensemble of KTX structures tightly converged with a coordinate precision of 0.6 Å and 1.3 Å for backbone and side chain heavy atoms, respectively. Backbone and most side chains had a well-defined orientation except the N- and C- terminal residues and Asn30 located in the loop connecting the second and third β-strand of KTX (Figure 3). The high-resolution solid-state structure of KTX deviates by 2.4 Å from the backbone conformation (PDB code: 1XSW) obtained on the basis of 28 manually assigned distance restraints[11], which deviates by 2.7 Å from the solution structure. The most pronounced deviation between 1XSW and the high-resolution structure was observed at the N-terminus, where four residues were rotated by about 50°, such that the first β-strand was straight and not bent as seen in the high-resolution structures (Figure S1).

Bottom Line:
Using an iterative assignment and structure calculation protocol, a large number of distance restraints was extracted from (1)H/(1)H mixing experiments recorded on a single uniformly labeled sample under magic angle spinning conditions.The calculated structure has a coordinate precision of 0.6 A and 1.3 A for the backbone and side chain heavy atoms, respectively, and deviates from the structure observed in solution.The approach is expected to be applicable to larger systems enabling the determination of high-resolution structures of amyloid or membrane proteins.

ABSTRACTHigh-resolution solid-state NMR spectroscopy can provide structural information of proteins that cannot be studied by X-ray crystallography or solution NMR spectroscopy. Here we demonstrate that it is possible to determine a protein structure by solid-state NMR to a resolution comparable to that by solution NMR. Using an iterative assignment and structure calculation protocol, a large number of distance restraints was extracted from (1)H/(1)H mixing experiments recorded on a single uniformly labeled sample under magic angle spinning conditions. The calculated structure has a coordinate precision of 0.6 A and 1.3 A for the backbone and side chain heavy atoms, respectively, and deviates from the structure observed in solution. The approach is expected to be applicable to larger systems enabling the determination of high-resolution structures of amyloid or membrane proteins.